Patient and sample collection
The retrospective study included 135 patients with confirmed diagnosis of COVID-19 who admitted to Beijing Ditan Hospital from January 20th, 2020 to April 27th, 2020. According to the guidelines on the diagnosis and treatment of new coronavirus pneumonia (version 7) released by National Health Commission of China, the classification of COVID-19 are as follows: Mild: Clinical symptoms from mild fever, respiratory tract to pneumonia manifestation. Severe: Meeting any one of the following should be treated as severe cases, including respiratory distress, respiratory rate ≥ 30 breaths/min; oxygen saturation ≤ 93% at rest; and PaO2/FiO2 ≤ 300. In severe group, 31 patients were admitted to ICU and 15 cases received mechanical ventilation. Twenty-five healthy donors matched to the age and sex of mild COVID-19 patients were enrolled. Three volunteers donated their peripheral neutrophils for in vitro experiments. Blood samples were collected by venipuncture into ethylenediaminetetraacetic acid tubes at the indicated timepoint and centrifuged at 450 × g for plasma separation. Samples were divided into small aliquots and stored at -80°C until the time of testing. The study was approved by Committee of Ethics at Beijing Ditan Hospital, Capital Medical University, Beijing, China. The approval number is JDLKZ(2020)D(036)-01.
Quantification of MPO-DNA and cfDNA
Cell-free DNA in plasma was quantified using the Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instruction. MPO-DNA complexes were quantified similarly to what has been previously described. In brief, a capture antibody against MPO was coated on a 96-well flat-bottom plate at 1:200 (Abcam, Cambridge, MA, USA), and the amount of MPO-bound DNA was quantified using the Quant-iT PicoGreen dsDNA assay as described above.
Quantification of C5 and C3
Plasma levels of C5 (including C5 and C5a) and C3 (including C3, C3a and C3b) were detected using Human Complement C5 ELISA Kit and Human Complement C3 ELISA Kit (Abcam) according to the manufacturer’s instruction.
NETs were detected by immunofluorescence staining as previously reported. Neutrophils were fixed, permeabilized and blocked after 3-hour in vitro culture or stimulation. Cells were incubated with antibody against histone H3 citrulline R2+R8+R17 (H3Cit) and followed by secondary antibody coupled with Alexa Fluor Dyes (Invitrogen). DNA was stained using 4’,6-diamidino-2-phenylindole (DAPI). Images were obtained with a confocal fluorescence microscope (Zeiss LSM 510 META; Carl Zeiss, Thornwood, NJ, USA). NETs were identified as structures positive for both histone H3Cit and DAPI staining.
Neutrophil isolation, in vitro culture and stimulation
Blood samples from healthy donors were collected into ethylenediaminetetraacetic acid tubes as described above for plasma separation. The anticoagulated blood was then fractionated by density-gradient centrifugation using Percoll (Stemcell Technologies, Vancouver, Canada). Neutrophils were further purified by dextran sedimentation of the red blood cell layer before lysing residual red blood cells with sodium chloride. Neutrophil preparations were at least 95% pure as confirmed by nuclear morphology.
Purified neutrophils were resuspended in RPMI-1640 medium supplemented with heat-inactivated 5% fetal bovine serum and 2 mM L-glutamine. Neutrophils were seeded into 96-well plate (5 × 104/well) for supernatant detection and 24-well plate (2 × 105/well) with polylysine-coated coverslips for NET immunofluorescence staining. Cells were rested for 1 hour at 37°C and 5% CO2 prior to COVID-19 plasma stimulation. Recombinant CPB (YaxinBio, Shanghai, China) was used to digest C3a and C5a in the plasma at 37°C for 30 min prior to neutrophil stimulation. Anti-human C3a antibody (Merck, Darmstadt, Germany) and anti-human C5a antibody (R&D Systems, Minneapolis, MN, USA) were added into the neutrophil culture system to neutralize C3a and C5a, respectively. Three hours later, the supernatant was collected to quantify MPO-DNA content. The results were calculated by deducting the background levels of MPO-DNA in the plasma.
Preparation of NET-conditioned medium and cell viability assay
Neutrophils from healthy donors were cultured with plasma from patients with COVID-19 as described above. Three hours later, the supernatant was collected carefully and used as NET-conditioned medium. HUVEC cells (3 × 103/well) were seeded into 96-well plate and cultured for 24 hours. Cells culture media were replaced with 100 μl NET-conditioned media or new cell culture media as control for 24 hours. Cell viability assay was performed using a cell counting kit 8 (CCK‐8) (Dojindo, Kumamoto, Japan) as per the manufacturer's protocol. Absorbance was detected at 590 nm using a microplate reader. The experiments were performed in sextuplicate.
All statistical analyses were performed with the SPSS 25.0 statistical package (IBM, Armonk, NY, USA). Values are presented as the mean ± standard deviation for data that were normally distributed or median and interquartile range for data that were not normally distributed for continuous variables and number (%) for categorical variables. The Kolmogorov-Smirnov test was used to inspect the normality and homogeneity of variance of all the data. For two-group comparison, P values were derived from the one-way Student t test to determine differences between groups with normally distributed data and Mann-Whitney nonparametric test with other data. For multi-group comparison, P values were derived from one-way ANOVA (continuous variables) or Chi-square test (categorical variables). For all comparisons, P < 0.05 was considered statistically signiﬁcant.